Hypothalamic Islands Research Articles

What really happens in the SCN at night

It is generally believed and clearly stated in most text books of physiology that the cells in the suprachiasmatic nucleus (SCN) of the hypothalamus show a daily variation in their activity and that this is reflected by changes in the spike frequency that can be recorded from the nucleus. Inouye & Kawamura (1979) showed that multiple unit activity recorded from hypothalamic islands increased during the time of day that the animals would have been inactive. A similar rhythm was reported in the activity of cells acutely dissociated from the suprachiasmatic nucleus (Welch et al. 1995) and in intact animals Meier et al. 1998). These studies are, however, incomplete in two respects; first, there is no analysis of the activity either to show that it is rhythmic in the statistically rigorous sense or to define and place confidence limits on the timing of the peaks and troughs of the activity. A new paper by Pennartz and colleagues in this issue of The Journal of Physiology does not address this issue but represents a great leap forward in another direction. They have shown, using rigorous whole-cell recording techniques, that although the number of cells in the SCN which respond to optic nerve stimulation stays constant, there is a difference in the post-stimulus whole-cell membrane current evoked by optic nerve stimulation at different times of day. During the day, the current mediated by the NMDA component of the EPSC in randomly selected cells in the SCN was found less frequently than it was during the night and when it was encountered, its magnitude was significantly smaller in amplitude (see Fig. 1). It is thus likely that despite its relatively low amplitude and slow time course, the NMDA receptor-mediated EPSC might exert a physiological role in modulating the retinal input to the SCN and thus controlling the activity of the cells which represent the circadian oscillator. Figure 1 Diagram to illustrate that, during the day, the SCN cells show only a short-duration current in response to optic nerve stimulation (long thin arrow). During the night, however, an additional current blocked by AP5 can also be detected (short broad arrow). ... This finding represents an important addition to our understanding of the daily modulation of the activity of the SCN. A great deal is known of the neurochemical changes which occur in the nucleus at different times of day (see e.g. Inouye & Shibata, 1994). Much recent work on circadian biology in mammals has been directed to determining the interrelation between the mouse equivalents (mPer 1, 2 and 3) of the Drosophila clock gene period (per) and the regulation of their activation by the protein products of another gene, Bmal1, leading to peaks in mPer1 transcription in the day and peaks of Bmal1 transcription at night (see e.g. Dunlap, 1999). Such changes alone do not affect either the activity of the SCN cells or their output. To affect the other regions of the CNS to which SCN cells project, changes in gene transcription must modulate spike intervals. Spike interval coding determines the release of transmitter from axon terminals and this in turn must depend on channel activity and membrane currents. The paper by Pennartz et al. (2001), while it does not establish a direct connection between, for example, the peak in Bmal1 transcription that occurs at night and the expression of the relevant receptor protein, is the first that characterises an important change in the activity of a specific receptor protein. Such changes almost certainly underpin the changes which are known to occur in the activity of the nucleus. It implies that the properties of a given neural pathway in the CNS are not necessarily constant at different times in the light-dark cycle. Thus neural connections are not all hard wired but change quite rapidly with time. Synaptic plasticity (at least in some situations) thus appears to arise from daily alterations in postsynaptic receptor expression. As time goes on we expect to see other reports of associated changes in the activity of different cell surface proteins.

Circadian Locomotor Rhythms, but Not Photoperiodic Responses, Survive Surgical Isolation of the SCN in Hamsters

Surgical isolation of the suprachiasmatic nuclei (SCN) within a hypothalamic island is reported to produce loss of circadian rhythmicity. The results have been interpreted to indicate that SCN efferents are necessary for the expression of circadian rhythms. It is not clear, however, whether the loss of circadian rhythms in behavioral responses following SCN isolation is attributable to transection of efferents, to loss of cells within the island, or to gliosis produced by the knife cut. To explore this issue, we examined locomotor activity and gonadal state of male golden hamsters housed in constant darkness (DD, with a dim red light for maintenance) for at least 10 weeks following isolation of the SCN from the rest of the brain by cuts by means of a Halasz wire microknife. Brain sections were immunocytochemically stained for the peptides vasoactive intestinal polypeptide (VIP), vasopressin (VP) or neurophysin II (NP II), and neuropeptide Y (NPY) to localize the SCN and to assess its viability, and for glial fibrillary acidic protein (GFAP) to delimit the border of the knife cut. Experimental animals with VIP and VP/NP II immunoreactivity in the SCN within the island retained free-running locomotor rhythms following transection of SCN efferents. Animals with cuts that failed to sever SCN efferents, and sham-operated animals (in which the Halasz knife was lowered but not rotated), also maintained circadian rhythmicity. Hamsters sustaining severe damage to the SCN showed disrupted locomotor activity. In those hamsters that retained circadian locomotor rhythmicity following SCN isolation, gonads failed to regress in DD, demonstrating the absence of an appropriate photoperiodic response. The results suggest a multiplicity of SCN coupling mechanisms in the control of circadian rhythms.

Intrinsic synapses in the ventromedial nucleus of the hypothalamus: an ultrastructural study.

To test the ideas that neurons intrinsic to a hypothalamic region might participate in steroid hormone effects and that intrinsic synapses might have unusual morphological features, we deafferented the ventromedial nucleus of the hypothalamus (VMH) in ovariectomized female rats with a Halasz knife. Some rats were given estrogen; others were endocrine controls. The main result of this study was that a large number of synapses in VMH survived the circumscribing cut. In fact, they comprised between one-half and two-thirds the number counted in unoperated controls. These were apparently from neuron cell bodies intrinsic to the medial basal hypothalamic island. Their morphology was not clearly different from that expected from unoperated controls or from other brain regions. A significant estrogen effect, an increase in the number of axodendritic synapses per unit area as seen in animals without surgical transections, was not present in the deafferented groups. This suggests that the hormone effect is due to synapses on VMH neurons from cell groups outside the medial basal hypothalamic island. If functional effects of estrogen mediated through VMH do not rely on intrinsic neurons, they must use estrogen-concentrating neurons that project to other brain regions.

Cholecystokinin: Induced suppression of feeding in fed, fasting and hypothalamic island rats

Experiments were undertaken to examine the involvement of CCK in the regulation of feeding behavior. The results indicate that 1) intact normal animals exhibited rhythmic circadian feeding behavior, with onset of feedings starting about two hr prior to dark period and continuing until 1 to 2 hr prior to the light period; 2) food deprivation and surgical isolation of the hypothalamus alter this pattern of feeding behavior, to the point that animals spend more time eating with no identifiable pattern; 3) administration of CCK to either fed, fasted or hypothalamic island animals has a profound dose related effect on suppressing feeding behavior and in modifying the eating pattern over 24 hr. A possible involvement of CCK with the opiate system in controlling feeding behavior is discussed.

GRF Neurons in the rat hypothalamus

The growth hormone-releasing factor (GRF)-containing neuronal system was immunohistochemically studied in the rat hypothalamus. The immunolabeled cell bodies were determined by intraventricular administration of colchicine 24 h before killing. In intact animals, the neurons appeared in the ventral portion of the arcuate nucleus (group 1) and in the area surrounding the ventromedial nucleus (group 2). Most of the cell bodies also indicated immunoreactivity for tyrosine hydroxylase (TH). The immunoreactive fibers accumulated showing a palisade arrangement in the external layer of the median eminence. The rats treated neonatally with monosodium glutamate revealed group 2 neurons and a few immunoreactive fibers in the median eminence. Half-anterolateral deafferentation of the medial basal hypothalamus, which was performed to isolate group 1 neurons or both group 1 and 2 neurons from the other brain parts, did not remarkably affect the appearance of the fibers in the median eminence. However, the perikarya were hypertrophic and strongly immunolabeled for GRF and TH. It is concluded that the fibers containing GRF in the median eminence derive mostly from group 1 neurons, and that the neurons may be regulated by an inhibitory mechanism by other neurons on the outside of the deafferented hypothalamic islands. GRF synthesized in group 2 neurons may act on other nneurons as a neurotransmitter-like substance.

Plasma thyrotrophin (TSH) response to intrahypothalamic injection of an alpha 2-adrenergic receptor agonist (clonidine).

To delineate the site at which the central noradrenergic (NE) system may exert its stimulatory influence on the thyrotrophin (TSH) secretion, plasma TSH concentrations were measured before and after clonidine (an alpha 2-agonist) administration to the hypothalamus of unanaesthetized, unrestrained rats. When the animals received a simultaneous injection of clonidine (0.2 microgram) and phenoxybenzamine (2.0 micrograms) into the dorsomedial nucleus (DMN)/the paraventricular nucleus (PVN), a significant rise in TSH levels was observed. Injection of the mixture into the anterior hypothalamus, posterior hypothalamus, ventromedial hypothalamus, and arcuate nucleus had no influence on the plasma TSH concentration. Clonidine (10.0 micrograms/100 g body weight) and phenoxybenzamine (100 micrograms/100 g body weight) injected into the jugular vein also caused a prompt increase in the plasma TSH concentration. In rats with an intact small hypothalamic island, the TSH response to the iv injection of the drugs was considerably diminished. These data support the view that NE receptors in the DMN/PVN are of importance in exerting an NE-stimulatory influence on the TSH secretion, probably through the TRH neurons.

Dissociation of circadian rhythms in rats with a hypothalamic island

Complete isolation of the medial basal hypothalamus, including the suprachiasmatic nuclei in the isolated island, from the rest of the central nervous system was performed in rats. The circadian rhythm of plasma corticosterone level remained essentially intact in 5 of 16 rats with complete islands, whereas that of spontaneous locomotor activity was decomposed into ultradian bursts in 15 rats, resulting in a clear dissociation of the two rhythms in four rats. One rat, whose circadian rhythm of both variables persisted after the hypothalamic isolation, showed a diurnal activity. The 24-h patterns of plasma corticosterone of the other rats could be characterized as either episodic or continuously low throughout the day. A prefeeding corticosterone peak was detected under restricted feeding in rats with episodic fluctuations but not in those with continuously low hormone levels. It is concluded that the hypothalamic island includes the fundamental structures necessary for the manifestation of the entrained circadian rhythm of plasma corticosterone in rats under ad libitum feeding, whereas it contains only a part of that for spontaneous locomotor activity. The coupling pathways from the circadian oscillator(s) to these overt rhythms seem to be separate in the central nervous system, and the circadian rhythm of plasma corticosterone is not a direct consequence of that of locomotor activity.

Neuroendocrine control of adrenocortical ornithine decarboxylase activity.

The increase in activity of adrenocortical ornithine decarboxylase (ODC) elicited by the administration of apomorphine (APM) was studied in rats, four days after transection of the spinal cord or 24 h after various types of brain surgery: transection of the mesencephalon or of the diencephalon, or hypothalamic deafferentation (creation of a "hypothalamic island"). Section of the cord elevated endogenous adrenocortical ODC activity and potentiated the induction of the enzyme by APM. Incomplete sections of the brain at the level of the mesencephalon or diencephalon produced no change in either endogenous or induced ODC activity. In contrast to this, interruption of the mesencephalic-diencephalic connections by complete diencephalic transection produced profound decreases of endogenous ODC and of the response to APM. Deafferentation of the hypothalamus raised endogenous ODC concentrations of the adrenal cortex and potentiated the response to APM. The results strongly suggest that APM acts at the level of the diencephalon (hypothalamus) to increase adrenocortical ODC activity. Diencephalic-mesencephalic connections must be intact for this to occur. Peripheral and extrahypothalamic influences play a modulatory role in this effect.

Learning takes place in rats with deafferentation of the medial basal hypothalamus

The present experiments were designed primarily to determine whether or not the neural connections of the hypophysiotrophic region of the hypothalamus are essential for learning (habituation, acquisition, retention, and extinction) in the male rat. The paradigms employed measured learning of a food-related habit, inhibitory and passive shock avoidance, and swim escape. In addition, measurements were made of body weights throughout the entire experimental period, of open field activity, of deposition of fecal boli, and of male sexual behavior. The results showed that learning took place approximately to the same extent in rats whether or not the hypophysiotrophic region was isolated from the rest of the brain. Thus, if the operation of the hypophysiotrophic region is essential at all for learning, humoral communication to and from the isolated region may be sufficient to maintain the required processes at a normal level. When placed on an ad libitum food intake, the hypothalamic island animals became obese. Impairment in male sexual function also was noted. In the feeding paradigm and five of seven measurements made in the other behavioral studies, the variabilities (standard deviations) were less among the animals with hypothalamic islands than in the operated controls. In measurements of the feeding habit, the variability in response rates among individual rats in the hypothalamic island group was significantly lower than among sham-operated animals during the times when high response rates were observed. One of the roles of the hypothalamus is to help maintain homeostatic constancy. It is suggested that another role, largely mediated by the activities of the hypophysiotrophic region, might be to emit neural and humoral signals that increase the number of behavioral options available to an individual organism, concomitantly increasing the variability among individuals, viz., the hypophysiotrophic region may be viewed as a variability generator. Increases in behavioral variability among members of a particular species in response to increasing environmental exigencies may enhance the ability of that species to survive.

Characteristics of a circadian pacemaker in the suprachiasmatic nucleus

The nature of the circadian rhythms of the SCN in a hypothalamic island was examined in male rats by recording multiple unit activity from the SCN for longer durations. Successful continuous recording lasted up to 35 days. Neural activity of the SCN inside the island showed free-running rhythms whose periods were slightly longer than 24 h (Figs. 2, 3, Table 1). When the retino-hypothalamic pathway was spared, re-entrainment to a displaced light and dark cycle was attained following a transition period of a few days (Fig. 4). Phases of the rhythms shifted in a phase-dependent manner in response to single light pulses interrupting constant darkness (Fig. 5 and Fig. 6). These results suggest an endogenous nature of the circadian rhythm of the SCN within the hypothalamic island. Thus, neurons or neuronal networks in the SCN may have not only an inherent ability to generate a circadian rhythm, but also an intricate machinery to regulate its phase. Simultaneous recordings from the left and right SCN showed a slight but visible discrepancy in their phases between the two rhythms in 3 out of 12 cases (Fig. 7).

Dopaminergic and adrenergic effects on plasma LH levels in rats with hypothalamic islands.

In female rats hypothalamic islands were prepared by making a cut around the mediobasal hypothalamus. Three weeks later the rats were ovariectomized. Two weeks after ovariectomy, part of the animals showed elevated blood LH levels with oscillations, although the mean LH levels were significantly lower than in sham-operated ovariectomized animals. There was a significant correlation between the occurrence of elevated blood LH levels after ovariectomy and the inclusion of at least part of the suprachiasmatic nucleus within the islands. Nor-adrenaline content of the hypothalamus was decreased significantly, but there was no significant change in hypothalamic dopamine content. Injection of doses of apomorphine, ranging form 2.5 microgram/kg to 25 mg/kg sc into island-bearing and sham-operated animals caused a dose-dependent decrease of plasma LH. However, the magnitude of the decrease was smaller in rats with hypothalamic islands than in controls. Even the highest doses of apomorphine did not decrease plasma LH further than to about 100 ng of LH-RP-1/ml. Phenoxybenzamine (10 and 40 mg/kg ip) and phentolamine (20 mg/kg ip) caused a decrease of plasma LH in ovariectomized control rats, but in ovariectomized rats with hypothalamic islands these drugs caused an increase. Clonidine (100 microgram/kg sc followed by 100 microgram/kg ip) had no effect in ovariectomized control rats, but in ovariectomized rats with a hypothalamic island an increase was induced. These results may have been caused by an increased sensitivity of hypothalamic alpha-receptors resulting from severing afferent noradrenergic fibres.

Distribution of β-lipotropin (β-LPH), adrenocorticotropin (ACTH) and α-melanocyte-stimulating hormone (α-MSH) in the rat brain. I. Origin of the extrahypothalamic fibers

By immunocytochemical techniques, the neuronal cell bodies containing ACTH, β-LPH and α-MSH have only been found in the area of the arcuate nucleus of the hypothalamus, whereas positive nerve fibers have been observed in many hypothalamic and extra-hypothalamic areas. The possible contribution of neurons which could be located in other brain areas has been studied in the rat by experiments involving hypothalamic deafferentation or destruction of the acurate nucleus. Fourteen days after deafferentation, no immunoreactive fibers could be detected in extrahypothalamic areas whereas the concentration of positive cell bodies and fibers remained unchanged within the hypothalamic island. In rats which had been injected in the neonatal period with monosodium glutamate (MSG), which selectively destroys the arcuate nucleus, only a few immunostained cell bodies were observed in hypothalamic region lateral to the arcuate nucleus. As compared to control animals, the concentration of immunostained fibers in both hypothalamic and extrahypothalamic regions was markedly decreased. These results strongly suggest that neuronal cell bodies producing ACTH, β-LPH and α-MSH are located in the region of the arcuate nucleus and send axonal projections into many brain areas.

Location and characterization of opiate receptors regulating pituitary secretion

The site at which opiate agonists and antagonists act to alter secretion of prolactin, growth hormone and luteinizing hormone as well as the pharmacological specificity of the opiate receptors mediating these effects were examined in rats. Injection of β-endorphin but not a 10 fold higher dose of the non opiate peptide β-endorphin, increased release of prolactin and growth hormone in male rats while inhibiting luteinizing hormone release in ovariectomized, estrogen primed female rats. Prior treatment with naltrexone i.p. blocked these responses. Injection of naltrexone into the hypothalamus lowered prolactin release. In rats with a surgically formed hypothalamic island systemic administration of morphine or naltrexone altered prolactin release in the same manner as was observed in intact animals. In contrast no effects of β-endorphin or naltrexone were observed on the spontaneous secretion of prolactin in vitro . In addition β-endorphin did not alter the inhibition of prolactin release produced by apomorphine in vitro . The ED 50 for stimulation of prolactin release following intraventricular administration of β-endorphin or the synthetic enkephalin analog FK 33-824 was the same, approximately 0.1 ng/rat. However FK 33-824 at 0.2 ng/rat was able to produce much greater analgesia and catatonia than β-endorphin. The metabolism and distribution of β-endorphin was examined but did not account for these differential effects. These results indicate that opiate agonists and antagonists can act at the hypothalamic but not the anterior pituitary level to alter release of prolactin, growth hormone and luteinizing hormone. In addition the data suggest that the opiate receptors mediating release of prolactin may have a different pharmacological specificity from those involved with analgesia and catatonia.

Localization of the site of the haloperidol-induced, prolactin-mediated increase of dopamine turnover in the median eminence: Studies in rats with complete hypothalamic deafferentations

Complete hypothalamic deafferentations were made in male rats with a modified Halász knife to isolate tuberoinfundibular dopaminergic neurons from the rest of the brain. A radioenzymatic procedure was employed to quantify dopamine and norepinephrine concentrations in various regions of the hypothalamus. Dopamine concentrations were unaltered while norepinephrine concentrations were reduced 50% in the median eminence and hypothalamic island 16-33 days after surgery. Basal serum prolactin concentrations were unaltered in these rats but were elevated 16 hours after the injection of haloperidol and 1 hour after alpha-methyltyrosine. The isolation of tuberoinfundibular neurons from the rest of the brain did not alter the ability of haloperidol to increase the rate of alpha-methyltyrosine-induced decline of dopamine in the median eminence. These results indicate that the haloperidol-induced, prolactin-mediated increase of dopamine turnover in the median eminence results from a direct action of this hormone on neurons within the medial basal hypothalamus.

Failure of stalk-median eminence (SME) extract to suppress serum prolactin levels in rats with hypothalamic islands

Failure of stalk-median eminence (SME) extract to suppress serum prolactin levels in rats with hypothalamic islands

Horseradish peroxidase study of intact or deafferented infundibular complex in Coturnix quail

Horseradish peroxidase was injected in the dorsal part of the infundibular complex and demonstrated histochemically in brain sections. In intact quail, cells labelled with peroxidase reaction product were found in the hippocampus, septum, lobus paraolfactorius, dorsal thalamus, preoptic and suprachiasmatic areas. Neither extrahypothalamic nor suprachiasmatic labelling could be detected after neural isolation of the basal hypothalamus, whereas testes growth was still induced by environmental photostimulation. Therefore, the photosensitive and gonadostimulating capacities of the hypothalamic island do not seem to depend upon uncompleted deafferentation of the hypothalamus.

Ultrastructural Localization of Acetylcholinesterase in the Arcuate Nucleus and Median Eminence of the Rat

Biochemical, physiological, pharmacological, and more recently enzyme histo- chemical data have indicated that cholinergic circuits exist in the hypothalamus. Ultrastructural correlates of these pathways such as acetylcholinesterase (AchE) positive neurons in the arcuate nucleus (ARC) and stained terminals in the median eminence (ME) have yet to be described. Initial studies in our laboratories utilizing chemical lesioning and microdissection techniques coupled with microchemical and light microscopic enzyme histo- chemical studies suggested the existence of cholinergic neurons in the ARC which project to the ME (1). Furthermore, in adult male rats with Halasz deafferentations (hypothalamic islands composed primarily of the isolated ARC and the ME) choline acetyltransferase (ChAc) activity, a good marker for cholinergic neurons, was not significantly reduced in the ME and was only somewhat reduced in the ARC (2). Treatment of neonatal rats with high doses of monosodium 1-glutamate (MSG) results in a lesion largely restricted to the neurons of the ARC.

Sensory responsiveness of single cells in the medial septal nucleus in intact and hypothalamic-deafferented rats

The effects of sciatic, acoustic, and photic stimuli on spontaneous single cell activity were studied in the medial septal nucleus in intact rats and in animals with chronic bilateral lesions of the medial forebrain bundle, complete hypothalamic deafferentations, and small posterior hypothalamic deafferentations. The changes in the rate and pattern of unit firing were analyzed statistically and the responsiveness compared between the intact rats and those with lesions. The spontaneous activity of about one-half of the units was affected by the sensory stimulation and there was a convergence of more than one modality on the same neuron. Units which had a lower rate of firing were facilitated and those which had a higher rate of firing were inhibited by sensory stimulation. The marked changes in the rate of firing were not accompanied by alterations in pattern of discharge. In rats with hypothalamic islands the mean spontaneous firing rate was increased; however, the percentage of the responsive cells and the degree of convergence were significantly reduced. The present data demonstrate extensive sensory projections and convergence in the septum, the transmission of the responses by the mediobasal hypothalamus, and modulation by the latter of septal electrical activity.

Adrenocortical responses to olfactory stimulation in rats with hypothalamic islands.

Department of Neurology, Hadassah University Hospital, Jerusalem, Israel (Received 27 October 1975) There exists both anatomical and electrophysiological evidence that olfactory pathways project to the hypothalamus (Scott & Pfaffmann, 1967; Scott & Chafin, 1975). Hypothalamic island preparations were used to differentiate between 'systemic' stresses which produced normal adrenocortical responses and 'neurogenic' stresses which caused a reduced adrenal discharge (Feldman, Conforti, Chowers & Davidson, 1970; Feldman, Conforti & Chowers, 1975). This paper describes the role of the hypothalamic neural connexions in the mediation of the adrenocortical response following olfactory stimulation. Experiments were performed on male albino rats of the Hebrew University strain weighing approximately 250 g. Adrenocortical responses to ether stress and to olfactory stimulation, as determined by changes in plasma corticosterone levels (Click, von Redlich & Levine, 1964), were studied in 33 intact animals and in 33 rats with hypothalamic islands (Halasz & Pupp, 1965). The protocol consisted of submitting

Adrenocortical responses following sciatic nerve stimulation in rats with partial hypothalamic deafferentations.

Studies from this laboratory have demonstrated that the adrenocortical response following sciatic nerve stimulation is completely inhibited in rats with hypothalamic islands, indicating that this response depends entirely on the activation of afferent neural pathways to the hypothalamus. With the purpose of identifying the site of entry of these neural pathways into the mediobasal hypothalamus the effects of partial hypothalamic deafferentations were studied. It was found that in rats with posterior or posterolateral deafferentation the adrenocortical responses were similar to those obtained in intact rats, while anterior and anterolateral deafferentation resulted in a reduction of 62.3 and 53.3%, respectively. These results would indicate that the sciatic impulses which activate the adrenocortical response involve neural afferents which enter the mediobasal hypothalamus by an anterior pathway.